To create a human therapeutic vaccine able to circumvent self-tolerance against tumor necrosis factor (TNF) alpha, foreign T helper epitopes were inserted into human TNFalpha, with minimal effect on the native three-dimensional structure. TNFalpha variants were screened for solubility, structural stability, biological activity, and after immunization, for eliciting inhibitory antibodies. The longest and most flexible loop in TNFalpha, also designated loop 3, is the only region that is not involved in intra- or intermolecular interactions and therefore constitute an attractive insertion site. However, the extension of the flexible loop by epitope insertions destabilized the TNFalpha molecule. Therefore, two cysteines were introduced to form a stabilizing disulfide bond between loops 2 and 3. In a second design approach, three TNFalpha monomers were linked by two T cell epitopes and expressed as a single chain TNFalpha trimer. TNFalpha variants that were expressed as soluble proteins also had a conserved tertiary structure, as determined by circular dichroism. The biological activity of the TNFalpha variants was of the same magnitude as human TNFalpha in cellular assays. Introduction of three separate single-point mutations (D143N, A145R, or Y87S) diminished the cytotoxicity of the mutated variants 50-800-fold compared with native TNFalpha. Antisera from mice immunized with the different TNFalpha variants were able to cross-react with native human TNFalpha and to inhibit TNFalpha signaling via the TNF receptors in vitro, suggesting that the structural binding epitopes of native human TNFalpha and thus the native conformation were conserved in the constructed vaccine candidates.